Mitochondrial Genome Diversity in the Central Siberian Plateau with Particular

Home , Baikal Mountains, Central Siberian Plateau, Fennoscandia, Ket people, North Asia, Tofalar

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Reference to Prehistory of Northernmost Eurasia

S. V. Dryomov*,1, A. M. Nazhmidenova*,1, E. B. Starikovskaya*,1, S. A. Shalaurova1,

N. Rohland2, S. Mallick2,3,4, R. Bernardos2, A. P. Derevianko5, D. Reich2,3,4, R.

I. Sukernik1.

1 Laboratory of Human Molecular Genetics, Institute of Molecular and Cellular

Biology, SBRAS, Novosibirsk, Russian Federation

2 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

3 Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA

4 Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA

5 Institute of Archaeology and Ethnography, SBRAS, Novosibirsk, Russian

Federation

Corresponding author: Rem Sukernik ([email protected])

* These authors contributed equally to this work.

Abstract

The Central Siberian Plateau was last geographic area in Eurasia to become habitable

by modern humans after the Last Glacial Maximum (LGM). Through comprehensive

mitochondrial DNA genomes retained in indigenous Siberian populations, the Ket,

Tofalar, and Todzhi - we explored genetic links between the Yenisei-Sayan region and

Northeast Eurasia over the last 10,000 years. Accordingly, we generated 218 new

complete mtDNA sequences and placed them into compound phylogenies along with

7 newly obtained and 70 published ancient mt genomes. Our findings reflect the origins

and expansion history of mtDNA lineages that evolved in South-Central Siberia, as

well as multiple phases of connections between this region and distant parts of Eurasia.

Our result illustrates the importance of jointly sampling modern and prehistoric

specimens to fully measure the past genetic diversity and to reconstruct the process of

peopling of the high latitudes of the Siberian subcontinent.

Although modern humans began colonizing sub-arctic and arctic Siberia 45,000

years ago, most archaeological sites postdate 12 kya, with the exception of episodic

incursions to the north of Eurasia during the warm phases, with the settlements primary

limited to cryptic refugia both in eastern Europe and Asia (reviewed by Pitulko et al.,

2017 [1]). During the last millennium, the Central Siberian Plateau, bounded by the

Yenisei River to the west, the North Siberian Lowland to the north, the Verkhoynsk

Mountains to the east, and the Sayan Mountains and Lake Baikal to the south, has been

shaped by key episodes that have had major impacts on human migrations, admixture,

and population replacement. At the end of the 15th century, when Russians first crossed

the Ural Mountains in substantial numbers, dozens of indigenous tribes and bands with

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markedly different ways of life and subsistence were widespread throughout Siberia

[2,3]. Soon after, some of these groups became extinct or underwent marked

postcontact decimation and admixture while others only diminished in number, their

territories shrinking to small patches of their former distributions. Two strata are

recognized in the aboriginal Siberian population: an ancient layer and a comparatively

new one, or the “Old Siberians” versus “Neosiberians” [4,5]. Most of the “Old

Siberians” who spoke languages belonging to linguistic outliers, such as Omok (an

extinct Yukaghir language spoken as late as the 18th century in the lower Kolyma and

Indigirka valleys), have been greatly reduced in number, and their cultures have been

brought under the domination of the more populous Neosiberians: Tungusic, Turkic or

Mongolian tribes, all relative newcomers to the region [2,3,5,6]. Nonetheless, groups

of Old Siberians still persist in remote “pockets” of Siberia in the form of

anthropological isolates, usually representing the now-amalgamating remnants of

small, previously distinct tribes and lineages. The study of mitochondrial DNA

variation to ascertain their origin and affinities, as well as their relationship to the first

Americans, has been a subject of intensive study since the early 1990s [7-9]. Candidate

founding lineages for Native American mtDNA haplogroups were identified by

identifying similar haplogroups in Eurasia [10-14]. For instance, unique mtDNA

sequences (haplogroups B4b1a, D4h2, and C1a) linked to Native American B2, D4h3a,

and C1, respectively, were attested using complete mtDNA sequences [10-12,14]. A

complete catalogue of mtDNA variation in the “Old-Siberians” is therefore important

not just for understanding the specific history of Siberia, but also the ancestry of peoples

of the New World.

A central concern of this study is disentangling ancestries from different sources

that mixed at different time points in the past to provide a better understanding of the

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genetic interactions between and within Siberian populations. Accordingly, we focused

on mitochondrial DNA genome diversity in the Yenisei-Sayan autochthonous

populations, primarily the Ket, Tofalar, and Todzhi (Fig. 1). Ket are now the sole

surviving member of the Yeniseian language family, speaking a unique language that

does not easily fit into any known phyla and is unrelated to any other Siberian language

[15]. Their related tribes, the Assan, Arin, and Kott, disappeared about 200 years after

their first contact with Russians. In this region, the Ket is the last tribe to retain their

original language and until recent times subsisted entirely on hunting, fishing, and the

gathering of wild plants [2,16,17]. The Tofalar and Todzhi languages whose members

originally spoke Samoyed belong to the subgroup of Turkic languages confined to the

upper Yenisei area [18-20], where they may have had ample opportunity to exchange

genes with the Ket or related tribes (Lopatin, 1940 [5], and references therein). We

compared mitochondrial DNA present-day diversity from these groups with complete

mitochondrial genomes from ancient samples from the region and placed them into

combined genealogical trees. We used these updated genealogies to trace ancestral

relationships between populations sharing subhaplogroup-specific mutations. We used

the results to reconstruct expansions (preponderantly from south to north) as well as

contractions of populations, thus shedding new light on northeastern Eurasia's past.

Populations and samples

Ket - Formerly called Yenisei Ostyak (the census of 1897 recorded 988

persons), the Ket people are thought to be descendants of some of the earliest

inhabitants of Central Siberia, while all of their present-day neighbors seem to be

relative newcomers. In the 18th century part of the Ket were forcibly moved to the lands

between the Ob and Yenisei Rivers, where the Selkup belonging to the Samoyed group

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of the Uralic language family lived [2,5,16]. Until recently, there were ~500 Ket living

in a few riverside villages in the middle reaches of the Yenisey; as in the past, many

survive as seasonal hunters, trappers, and fishermen. The remnants of the Southern

(Upper) Ket, whose ancestors are believed to have originated from the Podkamennaya

Tunguska region, have been almost completely integrated into the expanding Evenki

[17].

We integrated our newly obtained mtDNA sequences from previously collected

DNA samples with previously published data [21] and collected new DNA samples

from indivduals residing in August of 2014 in the villages of Turukhansk and Kellog

(Turukhansk District, Krasnoyarsk Region, Russian Federation). The total Ket sample

subjected to complete mtDNA sequencing was 38 individuals. Based on self-reported

ancestry, verified by senior members of related families, we determined that the

overwhelming majority of the Ket have been extensively admixed with Russians. A

substantial proportion of the Ket were not completely certain of their maternal ancestry

in terms of Ket, Selkup, or Evenki origin.

Tofalar – Fewer than 400 Tofalar, who are reindeer breeders and hunters,

inhabit the northern slopes of the eastern Sayan Mountains, along the rivers Uda,

Gutara, and Nerkha. They originally spoke a Samoyed language, but later changed to a

language of the Turkic family. The Tofalar are comprised of the remnants of several

hunting tribes, gradually assimilated into a broader group. In the 17th century, the

predecessors of the Tofalar entered into five administrative settlements of "Udinsk

Land" of Krasnoyarsk Region [2]. When the Tofalar transitioned to a settled lifestyle

in 1930, they were concentrated in three newly established villages in the territory of

Nizhni Udinsk District encompassing Upper Gutara, Nerkha, and Alygdzher. There is

anthropological and linguistic evidence that classifies Tofalar among the ‘Old

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Siberians’ [5]. In this study, we revised the analysis of previously collected Tofalar

mtDNAs from Upper Gutara and Nerkha villages [10] and supplemented existing data

with new blood samples from the easterenmost village of Alygdzer collected in July

2015. The total Tofalar/Udinsk Buryat sample subjected to complete mtDNA

sequencing constituted 40 individuals.

Todzhi - This is a small subgroup living in Todzhinsky District in the northeast

part of the Tuva Republic encompassing the intermountain Todzhinsky Depression

between the Western and Eastern Sayan. Although modern Todzhi people speak a

dialect of the Tuvan language, anthropologically they are distinct from southern and

western Tuvans, being similar to the neighboring Tofalar people [18]. Census records

of 2002 noted about 3000 Todzhi, and their traditional economy is generally considered

to have had a hunting-gathering emphasis supplemented by reindeer herding and

fishing. In this study, we focused on 160 blood samples collected through fieldwork in

February 2017 in three main settlements of the Todzhinsky District (Toor-Khem, Adyr-

Kezhig, and Iy). We selected unrelated samples from elders (n=51) born in 1962 and/or

earlier for full mtDNA sequencing.

RESULTS

In what follows we describe the genetic diversity of complete mtDNA

sequences from the Ket, Tofalar, and Todzhi, and compare them with previously

reported data. We also combined this with mtDNAs from the Mansi, Tubalar,

Nganasan, Evenki, Even, Yukaghir, and Koryak, many of whom we sequenced to the

full mtDNA genome level, building on lesser amounts of data previously reported from

these samples. A brief description of each population, as well as details of the sampling

collection are reported in the work of Torroni et al. 1993b [8]; Derbeneva et al. 2002a,

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b [21,22]; Starikovskaya et al. 2005 [10]; Volodko et al. 2008 [11]; Sukernik et al. 2010,

2012 [12,23].

Ket in their Eurasian context

Haplogroup N2a

The Yenisei region was outside the main routes of Eurasian agricultural

exchange up to the time of the Late Bronze Age Karasuk Culture [24-26]. One of the

remarkable features of the Ket mtDNA pool is a lineage of hapogroup N2a

distinguished by a set of mutations that we newly document here (m.1633T>C,

m.11722C>T, and m.12192G>A) (Fig. 2). Whereas the previously published N2a

sample uniquely marked by m.10841A>G (EU787451) is from the Upper Ket in the

village of Sulamai on the Stony Tunguska River [21], our newly identified N2a

mitogenome lacks this mutation, and comes instead from the village of Kellog in the

Lower Ket. Apart from the Ket individuals, none of the other extant or ancient Siberian

populations sampled to date are known to carry N2a. This haplogroup is found in just

a few contemporary individuals from Europe, Iran, Arabia, and Ethiopia [27,28]. It is

likely that this lineage came to the mid-Yenisei from Caucasus, presumably the major

source of N2a, which contributed to the central Siberian maternal lineages.

Haplogroup U4a/U4a1

Haplogroup U mtDNAs, confined to subhaplogroups U2e1, U3, U4a, U4b, U4c,

U4d, U5a1, is well represented in Old Siberian groups in the northern Altai-lower Ob-

Yensei triangle [11,12,21,22,29] including in our newly reported data. We built a

phylogenetic tree selectively for U4a/U4a1 subhaplogroups, including ancient mtDNA

sequences and their Ket counterparts, or those from the regions close to the Ket

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traditional territory (Fig. 3). The entire tree, combining the Ket, Tubalar, Mansi, and

Vadei Nganasan sequences with ancient mt genomes, a newly reported genome from

Novosibirsk (I0992: 5002-4730 calBCE), and a newly reported genome from the

neighboring Kemerovo region (I2074:5602-5376 calBCE), allows us to suggest that the

U4a1 lineage was present by Neolithic times in Altai-Sayan, where it could have

diversified in situ, with U4a1 and U4a3 dispersing into the heartland of Europe.

Complete U4a1 mtDNA sequence in the Yamnaya (I0231) [30] culture (the Beaker

people of western Europe in the early Bronze Age represented the far western extent of

Yamnaya ancestry spread) from Samara region and Mesolithic genomes from Sweden

is consistent with the association of the U4a lineage in West Siberia with Mesolithic

ancestry.

Haplogroup U4d

The updated phylogeny of U4d includes both its main subhaplogroups U4d1

and U4d2, and three previously unreported sublineages documented in two Ket and two

Mansi mt genomes (Fig. 4). Whereas the U4d1 sequences are prevalent in the Baltic

Sea region, the U4d2 haplogroup is associated with various tribes in areas where eastern

Uralic-speakers [11] expanded. Specifically, the observation of U4d1 sequence in a

Bronze Age individual (RISE500) from Kytmanovo (along the Chumysh River in the

northwestern Altai) supports the hypothesis of an east-to-west pattern of divergence of

Uralic languages. The coalescent time of the U4d1 and U4d2 subclusters (~4.7 kya) is

consistent with the hypothesis of continuous gene flow between the Yenisei River

valley and the eastern Baltic region during and following the Bronze Age. This

conclusion is in agreement with ancient genome-wide data from Finland and the

Russian Kola Peninsula [31], revealing that the specific genetic makeup of northern

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Europe traces important ancestry from Siberia migration that began at least 3,500 years

ago.

Haplogroup U5a

Recent studies utilizing the genome-wide approach suggest that U5a lineages

already existed in Mesolithic Fennoscandia (U5a1 and U5a2) and may imply an eastern

origin for these sublineages in Europe [32,33]. Three of our new Ket sequences

clustered into U5a1d2 along with a newly reported ancient mt genome (I2068: 420-565

calCE) from the Yenisei-Sayan area, whereas two of the Tubalar sequences fall into

U5a1d2b. Previously unrecognized haplogroup U5a2 haplotypes (U5a2b and U5a2a1b)

were identified in a few Mansi mtDNAs, chosen from previously collected samples

[22]. With a novel U5a1h harbored by a Mansi individual, these findings revise our

understanding of U5a1 haplogroup relationships (Supplementary Table 1, Fig. 5).

Haplogroup A8

Through the course of this study, previously published and newly obtained

A8a2 samples were explored to redefine the structure of the entire A8 tree

(Supplementary Table 1; Fig. 6). As a result, we were able to delineate a previously

uncategorized A8b lineage [34]. The immediate split of A8 created a distinctive A8b

evident in two Koryak mt genomes. The entire tree expands our understanding of

haplogroup A8 by encompassing previously attested ancient mt genomes attributed to

the Bronze Age Okunevo culture in Khakassia. Hence, genetic and archeological

evidence support a single/common origin of a population directly related to the

maternal ancestors of some of the present-day Ket, Tofalar, Tuvan, Yakut, Buryat, as

well as the Koryak individuals. It has been suggested that the Okunevo Culture derived

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ancestry from long-resident populations of the Altai-Sayan Upland whose roots may

extend back to the Neolithic, if not before [35].

Mitochondrial DNA gene pool in Tofalar and Todzhi

The present-day Tofalar and Todzhi do not cluster with the Russian Buryat who

live just to the northeast ofMongolia. Unlike the Ket, the Tofalar and Todzhi showed

high frequencies (>50%) of the east Eurasian haplocluster C4a’b, a pattern observed in

our previous studies of the Tofalar and Evenki [10]. In most cases, the results initially

obtained by examination of a part of the C4a and C4b sequences have been confirmed

and extended by subsequent surveys based on complete mitochondrial DNA analysis

[11]. Furthermore, eight of the Tofalar/Udinsk Buryat sample (20%) harbored

mitogenomes assigned to haplogroup M8a. This lineage, as well as its main derivatives

(M8a1, M8a2, M8a3), has not been observed among circumpolar groups, being

confined to southern and southeastern Siberia (Supplementary Fig. 1). The geographic

origin of M8 root appears somewhere within South-East Asia, subsequently splitting

into M8a and CZ clusters in the area currently inhabited by modern East Asians [36].

Haplogroup C4

The age estimates of the newly described sublineages hint to the origin and

diversification of the C4 haplogroup in Siberia/Asia (Fig. 7). Thus, modern samples

(C4d and C4e) nested in C4-m.152T>C node together with the newly reported ancient

individual I2072 (Solontsy5 from Altai dating to 3959-3715 calBCE) and RISE602

samples (Sary-Bel, 9/700 BC – AD 500/1000). Likewise, I1000 from Obkhoy, from

the Glazkovskaya culture and dated to 2871-2497 calBCE, all clustering to C4, support

ancient gene flow within southern extent of the Central-Eastern Siberia. The

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coalescence time estimates of the C4-m.152T>C-m.16093T>C were estimated to be

around 20 kya. Unlike the C4e subhaplogroup revealed in northwestern Altai, the C4d

lineage found outside Siberia shared common ancestry with the Tibetans or populations

currently living with Tibetans [37,38]. On the other hand, C4c, a sister group of C4a

and C4b [11] is found solely in the Western Hemisphere. The C4c present-day

distribution in North America is centered on the southern end of the former glacial ice-

free corridor. If this is a significant signature of its early dispersal history, it could

plausibly have been carried by the earliest peoples of central North America, who are

argued on genetic [39] as well as archaeological and paleoecological grounds to have

expanded southward from Beringia/Siberia into Americas sometime before 13,500

years ago [40,41].

Haplogroups C4a1 and C4a2

The Tofalar show high frequencies (>50%) of haplogroup C4a, a pattern

observed in our previous study of the Tofalar, Udinsk Buryat, and Evenki [10]. In most

cases, the results initially inferred from RFLP-analysis and HVSI sequencing have been

confirmed and extended by subsequent studies (new and published) based on complete

mitochondrial DNA analysis [11,12,28, the study herein]. Thus, C4a1 haplotypes,

common in the Tofalar, Todzhi, Evenki, Even, and northern Altaian (Kumandin,

Chelkan, and Teleut) samples, when assembled into one phylogenetic tree with their

ancient counterparts, have redefined the ancestral C4a1 types. At its widest extent, the

carriers of C4a1 lineages and sublineages spread from the Himalayas, Mongolia, and

Iran across the south-central Siberia to Chukotka (Supplementary Fig. 2), contrasting

with C4a2 diversity which is basically restricted to C4a2a (Supplementary Fig. 3).

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Haplogroup C5

The phylogeny of C5 is structured into four major branches, assigned as

C5a, C5b, C5c, and C5d, in accord with the latest release of PhyloTree (Supplementary

Fig. 4-6). While the C5a1 and C5a2a samples are from the Altaic-speaking populations

scattered across the southern extent of Eastern Siberia, the C5a2b is likely to reflect

different migrations of Reindeer Koryak, Chukchi, Yukaghir, and Kamchatkan Even.

An updated C5-m.16093T>C network, including 29 entire sequences, of which 15 are

new, is given in Supplementary Fig. 4. Accordingly, the entire tree splits into two main

lineages, C5c and C5d. While the C5c-m.16291C>T lineage is confined largely to the

Teleut and Kumandin from southwestern Siberia, C5d1 is associated with the groups

spread in the central/northern area where the Tungus-speaking populations expanded.

In addition to Okunevo samples previously reported from West Siberia [42], ancient

samples of this type were attested in the TransBaikal; one is Mesolithic (irk00x) and

the other is pre-Bronze Age time depth (irk078) [43].

The tree of haplogroup C5b (Supplementary Fig. 6) is conspicuous for the

C5b1a1 sublineage harbored by 4 of 39 (10.3%) Nganasan, 4 of 40 (10.0%) Tofalar,

and 3 of 51 (5.9%) Todzhi individuals. The entire picture indicates a relatively recent

affinity of the Nganasan from the Taimyr Peninsula to the Todzhi and Tofalar people.

This finding is congruent with phylogenetic analysis of autosomal HLA class II genes

in the respective populations. On all the neighbor-joining trees based on gene

frequencies at HLA class II loci, the Nganasan cluster together with the Tofalar and

Todzhi populations [43,45], thus corroborating a close genetic link between reindeer

hunters of the Taimyr Peninsula and reindeer breeders of the eastern Sayan Mountains.

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Haplogroup Z1

A novel Z1 sublineage, designated here as Z1b, and formed by two Tofalar

samples, with one haplotype from western Sayan [10] and the other from eastern Sayan

(this study) is noteworthy (Supplementary Fig. 7). An ancient sequence falling within

the same sublineage of Z1, harboring new m.9804G>A, m.10295A>G, and

m.15172G>A, was identified in the Korchugan site from the Novosibirk region, which

must have arrived in Central Siberia by the Neolithic, recovering it from an individual

dated to 5206-4805 calBCE (I0991). We also document an instance of Z1a1 in the

nearly reported ancient sample I0998 (Khuzhir site from Olkhon Island in Lake Baikal,

2835-2472 calBCE). An entire tree features Z1 coalescing at ~11.8 kya, whereas the

Z1a node is much younger, ~4.9 kya. Distinct sub-lineages of the Z1a haplogroup and

their derivative haplotypes harbored by the Tubalar, Tofalar, Nganasan, Yukaghir,

Koryak and Tungusic-speaking Evenki, Even, and Ulchi, could be a major part of

Neolithic dispersals from southeastern Siberia, with Z1a1a emerged in the present-day

Ket, Volga-Urals, and Finns and Saami much more recently. Occurrence of the Z1a1b

haplotypes in single carriers, referred to previously as Nganasan or Yukaghir [11,12],

in fact may be of Tungusic origin due to relatively recent gene flow from nearby Evenki

or Even groups that patchily inhabited Arctic Siberia since early the period of Russian

expansion.

Haplogroup F1b1

Of the 51 mt genomes sampled from Todzhi, who have long inhabited the refuge

between western and estern Sayan Mountains, the territory of presumptive origin of

some of the Ket’s ancestors, 8 individuals (15.7%) fall into two different sublineages

of the F1b1b haplogroup (Supplementary Table 1, Supplementary Fig. 8). Accordingly,

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the phylogeny of F1b1b is structured into three major sub-branches, assigned as

F1b1b1, F1b1b2, and F1b1b3. It is not surprising to observe the Ket identity of F1b1b2

confirmed by sharing of m.10227T>C and m.16179C>T variant, revealed earlier in

27.3% of Ket restricted to Podkamennaya Tungusska [21]. Remarkably, F1b1b2 was

attested in a Late Bronze Age individual from Afontova Gora on the left bank of the

Yenisei River (RISE553), and F1b1b3 in another, roughly contemporaneous sample

from the same site and archaeological culture (RISE554). Recently, older samples

(F1b1b) were attested with deeper, pre-Bronze Age time depth in the region west and

north of Baikal Lake (irk036, irk068) [43].

Discussion

Postglacial recolonization of Northeastern Siberia

An unresolved issue in the postglacial resettlement of the Siberian Arctic is

population substructure before the era of pastoralism, which emerged in the form of

reindeer herding after ~1200 CE [46] and could have had significant impacts on the

population-genetic landscape of this vast region. The Taimyr Peninsula appear to be

the last refuge for Nganasan, a palimpsest of reindeer hunters, the least touched

genetically by surrounding herding groups [11, 47-49]. By the mid-17th century, the

remnants of the westernmost Yukaghir tribe (Tavghi), the main ancestors of the

Nganasan, was fused with the Yenisei Samoyeds (Somatu tribe) giving rise to the

formation of the Avam Nganasan. A small subgroup of Yukagirized Tungus, later

Vadey Nganasan, was recorded only in 1897 [6]. As recently as the 1950s, the

Nganasan retained a lifestyle comparable to, and arguably continuous with that of pre-

pastoral reindeer-hunters [46,50,51].

A major complication in studying genetic prehistory of Yukaghir tribes is

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profound admixture with NeoSiberians, the Tungus-speaking populations and (to lesser

extent) the Yakut [11,52,53]. This admixture is a challenge for learning about the

historical relationships amongst the Yukaghir and surrounding populations within a

loose and poorly-defined North Siberian ethno-cultural complex composed of

linguistically heterogeneous groups that, at the beginning of the Russian exploration,

inhabited a region of northeastern Siberia, roughly between the Arctic Ocean in the

north, the Verkhoyansk range in the west and southwest, the Kolyma range in the

southeast, and the Pacific coast in the east [54, p.198]. To overcome this complication,

we restricted analyses to mtDNAs attributed to Yukaghir-specific haplogroups, to C4b

and D4b1c (D3), from adjoining groups, and to mtDNAs from more distant populations

without evidence of admixture. The pattern of the basal branches of C4b detected

among the modern and ancient inhabitants sampled from the Yana-Indigirka-Kolyma-

Anadyr interfluvial (Supplementary Fig. 9), suggest that the initial episodes of the C4b

diversification occurred in the Lena/Amga/Aldan area (south-central Yakutia) in the

Neolithic and was then dispersed by geographic expansion during the Bronze Age and

later. This conjecture is supported by an exceptionally large variety of C4b sublineages

sharing C4b root (m.3816A>G) in populations of different linguistic affiliation.

Interaction between reindeer Koryak, Chuvan, Khodyn and Anaul (the latter two tiny

tribes dissolved among the Chuvan (Chuvantsi) shortly after the first Russians appeared

in Chukotka in the mid-17th century) is consistent with historical records indicating that

quite a few of Yukaghir and Chuvantsi women were amongst the Koryak and the

Chukchi by the end of 19th century [6,55]. We also highlight the evolutionary history

of D4b1c/D3, whose intrinsic diversity has not yet been well resolved. Following

increased sampling, the haplogroup D4b1c (D3) phylogeny encompasses seven

Nganasan and four Yukaghir, which together account for 61.1% of the entire D3 sample

15 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

tested at the complete mtDNA level (Supplementary Table 1; Supplementary Fig. 10).

Furthermore, D4b1c/D3 in samples irk022 (2455-2200 calBCE) of Bronze Age from

Cis-Baikal, N5a (4340-4235 calBCE) of Middle Neolithic from Yakutia recently

determined by Kılınç et al. (2018) [43], serve as additional support for the

Nganasan/Yukaghir/Chuvan association. Co-representation of C4b and D3, being

highly confident in the Neolithic mtDNA genomes sampled from Yakutia [43], suggest

that the ancestry of the Yukaghir is multipartite and derived from the area that

encompassed the East Sayan, Lena/Aldan valleys, and the northernwestern vicinity of

Lake Baikal. The genetic continuity characterizing the ancestors of the Yukaghir was

apparently interrupted by the arrival of a new population who arrived from Siberia taiga

but whose original homeland was the in Trans-Baikal Mountains of north Manchuria

[56,57,54, p.198].

Conclusion

Here, we were assembled novel data on unique native Siberian populations, the

Ket, Todzhi, and Tofalar. The timing and whereabouts of Yenisei Ket population

history are long-standing issues [21,44,58]. The new mtDNA data provide a clearer

picture of the number and timing of founding lineages. Our findings appear to reflect

the origins and expansion history of mtDNA lineages that evolved in South-Central

Siberia bordering Central-Inner Asia, as well as multiple phases of connections between

this region and distant parts of Eurasia. Our results indicate that despite some level of

continuity between ancient groups and present-day populations, the entire region

exhibits a complex population history during the Holocene.

16 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Methods

Mitochondrial genome sequencing for modern samples

Genomic DNA was extracted from blood buffy coats using standard procedures.

The complete sequencing procedure of modern samples entailed PCR amplification of

2 overlapping mtDNA templates, which were sequenced with an Illumina HiSeq 2000

instrument [Illumina Human mtDNA Genome Guide 15037958B]. Short reads were

analyzed with the BWA-backtrack tool [59] and Unipro UGENE version 1.21 software

[60].

Ancient DNA Analysis

In a dedicated clean room at Harvard Medical School, we prepared powder from

the teeth of 7 individuals, all of whom we directly radiocarbon dated using accelerator

mass spectrometry. These individuals consisted of:

• I0991 (Korchugan1-3, Novosibirsk, Korchugan 1; 5206-4805 calBCE

(6060±50 BP, Poz-83427)),

• I0992 (Korchugan1-7, Novosibirsk, Korchugan 1; 5002-4730 calBCE

(5990±50 BP, Poz-83428)),

• I0998 (Khuzhir-2, Irkutsk, Olkhon Island, Lake Baikal, Khuzhir; 2835-2472

calBCE (4040±35 BP, Poz-83426)),

• I1000 (Obkhoy-7, Irkutsk, Kachugskiy, Obkhoy; 2871-2497 calBCE (4100±40

BP, Poz-83436)),

• I2068 (230/3, Kurgan 2, Sayan Mountain, Minusinskaya Intermountain Basin,

Tepsei III; 420-565 calCE (1560±30 BP, Poz-83507)),

• I2072 (230/13, Altai foothills, Solontcy-5; 3959-3715 calBCE (5050±40 BP,

Poz-83497)),

17 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

• I2074 (230/18, Burial 1, Vas'kovo 4, Intermountain basin between spurs of Altai

and Sayan Mountains; 5602-5376 calBCE (6520±40 BP, Poz-83514)).

Generation of the ancient DNA followed previously established methodology,

so we do not discuss them in detail here [61]. Briefly, we extracted DNA [62], and

converted it into individual barcoded double-stranded libraries in the presence of

Uracil-DNA Glycosylase (UDG) and Endo VIII (USER, New England Biolabs) to

characteristic errors at all but the final nucleotide [63]. We enriched the libraries for

sequences overlapping the mitochondrial genome [64], and then sequenced on an

Illumina NextSeq500 instrument using v.2 150 cycle kits for 2×76 cycles and 2×7

cycles. We computationally removed the barcode and adapter sequences, and merged

pairs of reads requiring a 15 base pair overlap (allowing up to one mismatch). We

mapped the merged sequeces to the reconstructed human mitochondrial DNA

consensus sequence [65] using bwa (v.0.6.1) [58], removed sequences with the same

strand orientation, start and stop positions, and built a consensus mitochondrial genome

sequence for each sample (average coverages were 25-1550-fold). We used

contamMix-1.0.9 to estimate 95% confidence intervals for the rate of mismatch to the

mitochondrial DNA consensus sequence, all of which were fully contained in the range

0.99-1 [66]. All samples had a rate of damage in the final nucleotide in the range of

0.028-0.127.

Mitochondrial data analysis

All mtDNA genome consensus sequences were called using SAMTOOLS

mpileup [67]. The resulting consensus sequences were then inspected by eye, with

particular attention being paid to the hypervariable regions and nucleotide positions

previously identified as being problematic [34]. All ambiguous sites were called as 'N'.

18 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Mitochondrial haplogroups were assigned with mitohg v.0.2.8 software [68].

Entire mtDNA sequences were assembled into phylogenetic trees by using

mtPhyl v5.003 software. Coalescence dates were estimated with the ρ statistic [69].

Standard errors (σ) were calculated according to Saillard et al. (2000) [70]. Mutational

distances were converted into years using the substitution rate for the entire molecule,

2.67×10−8 substitutions per site per year [68]. The haplogroup affiliations reported in

this analysis correspond to the current nomenclature of mtDNA in agreement with the

latest release (February 2016) of PhyloTree Build 17 [34].

Overall, 218 newly reported mt genomes are listed in Table S1 along with

ethnicity, sample location, and accession codes in GenBank. In the course of this study,

70 ancient mtDNA sequences were published and we used them to provide powerful

new information about subhaplogroup affiliation (Supplementary Table 2)

[24,25,30,33,41,43]. To uncover ancient mtDNA lineages, especially those related to

the Altai-Sayan area, we compared modern mitogenomic data with their ancient

counterparts sampled from skeletons recovered from the Euro-Siberian region that

extends from the Central Europe and Scandinavia to Lake Baikal.

The sequence data for the new mitogenomes (n = 218) are available in Genbank

with accession numbers MG660609-MG660750, MG778654-MG778683, MH807359,

MH807364, MK180566, MK180567, MK180570, MK180572, MK180574,

MK180577, MK180579, MK180580, MK217912-MK217947.

19 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

References

1. Pitulko, V., Pavlova, E. & Nikolskiy, P. Revising the archaeological record of the Upper

Pleistocene Arctic Siberia: Human dispersal and adaptations in MIS 3 and 2. Quat. Sci.

Rev. 165, 127–148 (2017).

2. Dolgikh, B. O. Rodovoi i plemennoi sostav narodov Sibiri v XVII veke. [The Clan and

Tribal Composition of Siberian Peoples in the 17th century] (in Russian). (Akademia

Nauk SSSR, 1960).

3. Levin, M. G. & Potapov, L. P. The Peoples of Siberia. (The University of Chicago Press,

1964).

4. Czaplicka, M. A. The Turks of Central Asia. (Clarendon Press, 1918).

5. Lopatin, I. A. The Extinct and Near-Extinct Tribes of Northeastern Asia as Compared with

the American Indian. Am. Antiq. 5, 202–208 (1940).

6. Jochelson, W. I. The Yukaghir and the Yukaghirized Tungus. The Jesup North Pacific

expedition, IX (1). (Memoirs of the AMNH) (E. J. Brill, 1910).

7. Torroni, A. et al. Asian affinities and continental radiation of the four founding Native

American mtDNAs. Am. J. Hum. Genet. 53, 563–590 (1993a).

8. Torroni, A. et al. mtDNA variation of aboriginal Siberians reveals distinct genetic

affinities with Native Americans. Am J Hum Genet 53, 591–608 (1993b).

9. Neel, J. V, Biggar, R. J. & Sukernik, R. I. Virologic and genetic studies relate Amerind

origins to the indigenous people of the Mongolia/Manchuria/southeastern Siberia region.

Proc. Natl. Acad. Sci. U. S. A. 91, 10737–41 (1994).

10. Starikovskaya, E. B. et al. Mitochondrial DNA diversity in indigenous populations of the

southern extent of Siberia, and the origins of Native American haplogroups. Ann. Hum.

Genet. 69, 67–89 (2005).

11. Volodko, N. V. et al. Mitochondrial Genome Diversity in Arctic Siberians, with Particular

Reference to the Evolutionary History of Beringia and Pleistocenic Peopling of the

Americas. Am. J. Hum. Genet. 82, 1084–1100 (2008).

20 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

12. Sukernik, R. I. et al. Mitochondrial genome diversity in the tubalar, even, and ulchi:

Contribution to prehistory of native siberians and their affinities to native americans. Am.

J. Phys. Anthropol. 148, 123–138 (2012).

13. Dryomov, S. V. et al. Mitochondrial genome diversity at the Bering Strait area highlights

prehistoric human migrations from Siberia to northern North America. Eur. J. Hum.

Genet. 23, 1399–1404 (2015).

14. Tackney, J. C. et al. Two contemporaneous mitogenomes from terminal Pleistocene

burials in eastern Beringia. Proc. Natl. Acad. Sci. 112, 13833–13838 (2015).

15. Ruhlen, M. The origin of the Na-Dene. Proc. Natl. Acad. Sci. U. S. A. 95, 13994–13996

(1998).

16. Dolgikh, B. O. Kety [The Kets] (in Russian). (OGIZ, 1934).

17. Alekseenko, E. A. Kety: istoriko-demograficheskie ocherki [The Kets: Historical-

ethnographic sketches] (in Russian). (Nauka, 1967).

18. Vainshtein, S. I. Tuvintsy-todzhintsy. Istorico-etnograficheskie ocherki [The Tuvan-Tozhu:

Historical-ethnographic sketches] (in Russian). (Nauka, 1961).

19. Potapov, L. P. Etnicheskiy sostav i proiskhozhdeniye Altaitsev [Ethnic composition and

origin of Altaians]. Istoriko-etnograficheskiy ocherk [Historical ethnographical essay] (in

Russian). (Nauka, 1969).

20. Rassadin, V. I. The ethnic composition of Tofalar. 30, (2014).

21. Derbeneva, O. A., Starikovskaia, E. B., Volod’ko, N. V, Wallace, D. C. & Sukernik, R. I.

Mitochondrial DNA variation in Kets and Nganasans and the early peoples of Northern

Eurasia. Genet. 38, 1554–1560 (2002a).

22. Derbeneva, O. A., Starikovskaya, E. B., Wallace, D. C. & Sukernik, R. I. Traces of early

Eurasians in the Mansi of northwest Siberia revealed by mitochondrial DNA analysis. Am.

J. Hum. Genet. 70, 1009–1014 (2002b).

23. Sukernik, R. I., Volodko, N. V., Mazunin, I. O., Eltsov, N. P. & Starikovskaya, E. B. The

genetic history of Russian old settlers of polar northeastern Siberia. Russ. J. Genet. 46,

1386–1394 (2010).

24. Allentoft, M. E. et al. Population genomics of Bronze Age Eurasia. Nature 522, 167–172

(2015).

21 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

25. Unterländer, M. et al. Ancestry and demography and descendants of Iron Age nomads of

the Eurasian Steppe. Nat. Commun. 8, (2017).

26. Juras, A. et al. Diverse origin of mitochondrial lineages in Iron Age Black Sea Scythians.

Sci. Rep. 7, 1–10 (2017).

27. Fernandes, V. et al. The Arabian cradle: Mitochondrial relicts of the first steps along the

Southern route out of Africa. Am. J. Hum. Genet. 90, 347–355 (2012).

28. Derenko, M. et al. Complete mitochondrial DNA diversity in Iranians. PLoS One 8,

(2013).

29. Naumova, O. I., Khaiat, S. S. & Rychkov, S. I. [Mitochondrial DNA diversity in Kazym

Khanty] (in Russian). Genetika 45, 857–861 (2009).

30. Mathieson, I. et al. Genome-wide patterns of selection in 230 ancient Eurasians. Nature

528, 499–503 (2015).

31. Lamnidis, T. C. et al. Ancient Fennoscandian genomes reveal origin and spread of

Siberian ancestry in Europe. Nat. Commun. 9, 5018 (2018).

32. Lazaridis, I. et al. Ancient human genomes suggest three ancestral populations for present-

day Europeans. Nature 513, 409–413 (2014).

33. Günther, T. et al. Population genomics of Mesolithic Scandinavia: Investigating early

postglacial migration routes and high-latitude adaptation. PLOS Biol. 16, e2003703

(2018).

34. van Oven, M. & Kayser, M. Updated comprehensive phylogenetic tree of global human

mitochondrial DNA variation. Hum. Mutat. 30, 386–394 (2009).

35. Jeong, C. et al. The genetic history of admixture across inner Eurasia. Nat. Ecol. Evol. 3,

966–976 (2019).

36. Marrero, P., Abu-Amero, K. K., Larruga, J. M. & Cabrera, V. M. Carriers of human

mitochondrial DNA macrohaplogroup M colonized India from southeastern Asia. BMC

Evol. Biol. 16, 1–13 (2016).

37. Zhao, M. et al. Mitochondrial genome evidence reveals successful Late Paleolithic

settlement on the Tibetan Plateau. Proc. Natl. Acad. Sci. 106, 21230–21235 (2009).

38. Qin, Z. et al. A mitochondrial revelation of early human migrations to the Tibetan Plateau

before and after the last glacial maximum. Am. J. Phys. Anthropol. 143, 555–569 (2010).

22 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

39. Kashani, B. H. et al. Mitochondrial haplogroup C4c: A rare lineage entering America

through the ice-free corridor? Am. J. Phys. Anthropol. 147, 35–39 (2012).

40. Potter, B. A. et al. Early colonization of Beringia and Northern North America:

Chronology, routes, and adaptive strategies. Quat. Int. 444, 36–55 (2017).

41. Braje, T. J., Rick, T. C., Dillehay, T. D., Erlandson, J. M. & Klein, R. G. Arrival routes of

first Americans uncertain-Response. Science 359, 1225 (2018).

42. Damgaard, P. de B. et al. The first horse hereders and the impact of early Bronze Age

steppe expansions into Asia. Science. 360, eaar7711 (2018).

43. Kılınç, G. M. et al. Investigating Holocene human population history in North Asia using

ancient mitogenomes. Sci. Rep. 8, 8969 (2018).

44. Uinuk-Ool, T. S., Takezaki, N., Derbeneva, O. A., Volodko, N. V & Sukernik, R. I.

Variation of HLA class II genes in the Nganasan and Ket, two aboriginal

Siberian populations. Eur. J. Immunogenet. Off. J. Br. Soc. Histocompat. Immunogenet.

31, 43–51 (2004).

45. Uinuk-Ool, T. S., Takezaki, N., Sukernik, R. I., Nagl, S. & Klein, J. Origin and affinities

of indigenous Siberian populations as revealed by HLA class II gene frequencies. Hum.

Genet. 110, 209–226 (2002).

46. Khlobystin, L. P. Drevnyaya istoriya Taimyrskogo Zapolyaria i voprosi formirovaniya

kul’tur Severa Evrazii [Ancient History of Taimyr and the Formation of the North

Eurasian Cultures]. (In Russian). (“Dmitry Bulanin”, 1998).

47. Dolgikh, B. O. Origin of the Nganasan. [Proishozhdenije nganasanov] (in Russian). Tr.

instituta Etnogr. im. N. Mikluho-Maklaja AN SSSR. 3, 5–87 (1952).

48. Gol’tsova, T. V & Sukernik, R. I. [Genetic structure of an isolated group of the indigenous

population of northern Siberia, the Nganasans (Tavginians) of Taimir] (in Russian).

Genetika 15, 734–744 (1979).

49. Karaphet, T. M., Sukernik, R. I., Osipova, L. P. & Simchenko, Y. B. Blood groups, serum

proteins, and red cell enzymes in the Nganasans (Tavghi)-reindeer hunters from Taimir

Peninsula. Am. J. Phys. Anthropol. 56, 139–145 (1981).

50. Simchenko, Y. B. Kultura okhotnikov na oleney severnoy Evrazii. [The culture of reindeer

hunters in northern Eurasia.] (in Russian). (Nauka, 1976).

23 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

51. Pitul’ko, V. Terminal Pleistocene—Early Holocene occupation in northeast Asia and the

Zhokhov assemblage. Quat. Sci. Rev. 20, 267–275 (2001).

52. Fedorova, S. A. et al. Autosomal and uniparental portraits of the native populations of

Sakha (Yakutia): Implications for the peopling of Northeast Eurasia. BMC Evol. Biol. 13,

127 (2013).

53. Duggan, A. T. et al. Investigating the prehistory of Tungusic peoples of Siberia and the

Amur-Ussuri region with complete mtDNA genome sequences and Y-chromosomal

markers. PLoS One 8, 1–19 (2013).

54. Zgusta, R. The Peoples of Northeast Asia through Time. Precolonial Ethnic and Cultural

Processes along the Coast between Hokkaido and the Bering Strait. (Brill Academic Pub,

2015).

55. Jochelson, W. The Koryak. Material Culture and Social Organization of the Koryak. A

publication of the Jesop North Pacific Expedition, X (2). (Memoir of the American

Museum of Natural History). (E. J. Brill, 1908).

56. Okladnikov, A. P. Istoriya Jakutskoj ASSR [A history of the Yakut ASSR]. (in Russian).

(AN SSSR, 1955).

57. Okladnikov, A. P. & Derevyanko, A. P. Dalekoye proshloye Primoriya i Priamuryia [The

distant past of Primorie and Priamurie] (in Russian). (Dalnevostochnoye knizhnoye

izdatel’stvo, 1973).

58. Flegontov, P. et al. Genomic study of the Ket: A Paleo-Eskimo-related

ethnic group with significant ancient North Eurasian ancestry. Sci. Rep. 6,

1–12 (2016).

59. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler

transform. Bioinformatics 26, 589–595 (2010).

60. Okonechnikov, K. et al. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics

28, 1166–1167 (2012).

61. Haak, W. et al. Massive migration from the steppe was a source for Indo-European

languages in Europe. Nature 522, 207–211 (2015).

24 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

62. Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave

bear reconstructed from ultrashort DNA fragments. Proc. Natl. Acad. Sci. 110, 15758–

15763 (2013).

63. Rohland, N., Harney, E., Mallick, S., Nordenfelt, S. & Reich, D. Partial uracil-DNA-

glycosylase treatment for screening of ancient DNA. Philos. Trans. R. Soc. B Biol. Sci.

370, 20130624–20130624 (2014).

64. Maricic, T., Whitten, M. & Paabo, S. Multiplexed DNA sequence capture of mitochondrial

genomes using PCR products. PLoS One 5, e14004 (2010).

65. Behar, D. M. et al. A ‘copernican’ reassessment of the human mitochondrial DNA tree

from its root. Am. J. Hum. Genet. 90, 675–684 (2012).

66. Fu, Q. et al. A revised timescale for human evolution based on ancient mitochondrial

genomes. Curr. Biol. 23, 553–559 (2013).

67. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25,

2078–2079 (2009).

68. Dryomov, S. V. mitohg v.2.0.8. (2019). Available at:

https://github.com/stasundr/gomitohg.

69. Forster, P., Harding, R., Torroni, A. & Bandelt, H.-J. Origin and evolution of Native

American mtDNA variation: a reappraisal. Am. J. Hum. Genet. 59, 935–45 (1996).

70. Saillard, J., Forster, P., Lynnerup, N., Bandelt, H.-J. & Nørby, S. mtDNA Variation among

Greenland Eskimos: The Edge of the Beringian Expansion. Am. J. Hum. Genet 67, 718–

726 (2000).

Competing interests: The authors declare no competing interests.

25 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure legends

Figure 1. Map of Siberia and adjacent part of Europe.

Black circles mark the settlements of sampling expeditions.

Black triangles denote locations of the ancient specimens generated through the course

of this study and listed in Supplementary Table 2.

General legend for phylogeny figures: In bold and green color is a new sequence

generated through the course of this study. When two or more identical samples belong

to the same group, their numbers are given in brackets. In bold and yellow are new

ancient sequences generated through the course of this study, in grey - ancient

sequences gleaned from published sources (see Supplementary Table 2). Dashed lines

for ancient samples indicate that the sequence is not complete and contain gaps due to

DNA damage or contamination.

26 bioRxiv preprint doi: https://doi.org/10.1101/656181; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 1. Map of Siberia and adjacent part of Europe.

60º 20º 80º 40º 60º 80º 100º 120º 140º 180º

60º

20º

Yenisey Turukhansk Ob

Kellog Lena

40º

Korchugan-1

40º Vas'kovo-4 V.Gutara Kushun Obkhoy Solontcy-5 Khuzhir Amur Tepsei-III Nerkha Iiy Alygdzher Toora-Hem 40º Adir-Kezhig

Figure 2. The phylogeny of haplogroup N2a complete sequences.

189 AG 709 GA 5046 GA 11674 CT 12414 TC

199 TC 195 TC 739 CT 204 TC 7581 TC 207 GA 15106 GA 1243 TC 16153 GA 3505 AG 16319 GA 5460 GA 8251 GA 8994 GA 11947 AG 15884 GC N2a 8.34 (5.11; 11.65) 16292 CT

W 14152 AG 4065 AG 189 GA! 1633 TC 16111 CT 8286 TC 11722 TC 8287dC 12192 GA N2a2 13074 AG N2a1 14743 AG

152 TC! 10841 AG 267 TC 3644 TC 198 CT 7269 GA 11914 GA 5261 GA 15628 AG 16086 TC 12007 GA 16293 AG

Persian Persian Persian Greek Armenian Armenian Ket Ket KC911573 KC911431 KC911368 Aromanian JF904935 JN381503 MG778664 EU787451 JN207845

Figure 3. The phylogeny of U4a distinct branches and subbranches.

U4a 8.66 (5.45; 11.95)

5655 TC 4688 TC 152 TC! 247 GA! 5705 AG 12937 AG 2792 AG 16113 AC 16134 CT 16362 TC

U4a1 5.41 (3.18; 7.68) 13105 AG! 6665 CT 6929 AG 8065 GA 16362 TC 13708 GA 72 TC 310 TC 198 CT 12950 AG 7153 TC 14635 TC U4a3 6617 CT U4a1d

801 AG 16265 AG 4206 AG 3460 GA 15773 GA U4a3a

16129 GA! 9335 CA

Karelia_HG Mansi Mesolithic Mesolithic Ket (8) Yamnaya_ Vadei Tubalar Tatar Novosibirsk_ Kemerovo_ Italian Czech Denmark Yuzhnyy FJ493507 SHG SHG MG778655 Samara Nganasan FJ147312 GU123034 Neolithic Neolithic EF060364 EU545423 JX153448 Oleni Ostrov Sweden Sweden MG778657 Ekaterinovka FJ493506 GU123031 Korchugan 1 Vas’kovo-4 Karelia SBj SF12 MG778658 I0231 I0992 I2074 I0211 MG778665 MG778667 MG778678 MG778660 MG778681

Figure 4. The phylogeny of U4d complete sequences; compared with a couple of ancient (incomplete) sequences.

U4d 10.76 (5.73; 16.00)

16240 AG 2772 CT 5567 TC 10692 CT 11326 CT 11518 GA 13105 AG! 16189 TC!

U4d1 4.79 (1.38; 8.32) U4d2 4.66 (2.01; 7.39)

3637 CA 16189 CT!! 1533 CT 5984 AG 13398 AG 10320 GA 16111 CT 1700 TC 6938 CT 16390 GA 11031 GA 8596 AG 12236 GA 152 TC! U4d1a U4d1b 12234 AG

8260 TC 15948 AG 3394 TC 1811 GA! 185 GA 189 AG 1193 TC U4d1a1

215 AG 16286 CG 13145 GA

Samara_ Andronovo Finnish Tatar Tatar Russian Estonian Estonian Avam Mansi (2) Ket Ket Tatar Mongolian Czech Russian Khakassian Eneolithic Kytmanovo subcluster GU123021 GU122982 EU545448 EST-92 EST-108 Nganasan MG660638 MG778674 MG778676 GU122988 EU007891 EU545426 KJ856699 KJ856735 Volga River RISE500 EST-41 Stoljarova FJ230891 MG660642 I0434 Stoljarova 2015 2015

Figure 5. The phylogeny of U5a1 revealing distinct branches or subbranches in the Ket, Tubalar, and Mansi mt genomes compared with ancient

sequences.

U5a1

3027 TC 150 CT 3192 CT U5a1d 12.34 (6.68; 18.26) 3591 GA 12618 GA 16239 CT 152 TC! 14053 AG 5263 CT 573.XC 13002 Ca 3552 TC 1303 GA 6895 AG U5a1d1 U5a1d2 4592 TC 13966 AG 11296 CT 16129 GA! 11938 CT 16192 TC!

241 AG 16051 AG 4823 TC 4924 Gc 7853 GA 10858 TC U5a1h 16093 TC 14110 TC 16172 TC 195 TC! 146 TC! 15217 GA 5583 CT 16304 TC 16189 TC! 16145 GA 16189 TC! 4215 AG U5a1d2b 14053 AG U5a1d2a

533 AG 16241 AG 6584 CT 16287 CT 6836 CT 16325 TC

U5a1d2a1

12406 GA 16092 TC

Mesoltihic Mesoltihic Karasuk Yamnaya Russian Pole Russian Belorussian Ket (3) Siberia_IA Tatar Tubalar (2) Yamnaya Denmark Denmark Mansi SHG SHG Khakassia Rostov region GU296612 GU296542 GU296599 GU296655 MG660620 Sayan Mount GU123032 FJ147317 Rostov region JX153519 JX154011 MG660643 Norway Norway RISE502 RISE240 MG660621 I2068 MG660604 RISE546 Steigen Hum2 MG778669

Figure 6. The phylogeny of A8 revealing distinct lineages or sublineages.

64 CT 5824 GA 146 TC! 12175 TC

A8a 10.87 (5.97; 15.96) A8b

151 CT 152 TC! 722 CT 253 CT 6962 GA 6779 AG 3144 AG 8865 GA 16293 AC 4508 CT 12777 AG 5046 GA A8a2 5067 AG A8a1 7711 TC 9531 AG 16291 CA 16242 TC! 199 TC 15796 TC 16193 CT 235 GA! 3912 AG 366 AG 16066 AG 15229 TC 7762 GA 16223 TC

9007 AG

Han Yakut Buryat Okunevo Okunevo Okunevo Tofalar Ket Ket Tuvan Okunevo Okunevo Koryak Chinese KF148096 EF153797 Uybat V Okunev Ulus Uybat III MG660738 AY519486 MG778662 MG660672 Verkhni Askiz Verkhni Askiz EU482364 FJ748724 KF148447 RISE681 RISE664 RISE677 RISE515 RISE667 EU482363 RISE670 RISE673

Figure 7. Schematic phylogeny of mtDNA haplocluster C4 sequences

2232.1 A 195 TC! 152 TC! 200 AG 204 TC C4a’b’c 8473 TC 16093 TC 151 CT 7307 AG 12672 AG 3816 AG 14433 CT 15479 TC 15148 GA 372 TC 3192 CT 4742 TC 7100 AG 7897 GA 15052 AG 16297 TC 12780 AG 15236 AG C4e C4a C4b C4c 12390 CT 13488 TC C4d 16566 GA 447 CT 4065 AG 8152 GA 14392 CT 5747dA 13641 TC 10314 CT 13722 AG 16129 GA 16223 TC 5978 AG 10248 TC 13581 TC 628 CT 195 TC! 5785 TC 6185 TC 6927 CT

Shamanka_EN Shamanka_EN Bronze Age Bronze Age Glazkovskaya Solontsy5_N Iron Age Tibetan Tibetan Tibetan Han Teleut Shor Cis-Baikal Cis-Baikal Cis-Baikal Cis-Baikal Irkutsk region Foothhills of the Sary-Bel GQ895156 GQ895155 GQ895165 Chinese MG660602 FJ951610 DA248 DA247 mak026 irk057 I1000 Altai RISE602 NA18631 DA249 I2072